Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor.
Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor.
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DOI：
10.1042/bj20120408
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发表时间：
2012-07-01
期刊：
影响因子：
--
通讯作者：
Pasquale EB
中科院分区：
文献类型：
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作者：
Lamberto I;Qin H;Noberini R;Premkumar L;Bourgin C;Riedl SJ;Song J;Pasquale EB
EphA4受体酪氨酸激酶与Ephin配体相互作用，调节许多过程，从轴突引导和神经再生到癌症恶性。因此，抑制EphA4结合的拮抗剂可能在各种研究和治疗应用中有用。在这里，我们表征了三个拮抗肽(KYL，APY和VTM)的结合特征，它们选择性地靶向EphA4。等温滴定量热法分析表明，这三种多肽都以较低的微摩尔亲和力结合到EphA4的Ephin结合域上。此外，一系列EphA4突变的影响表明，这些肽以不同的方式与EphA4的ePhin结合口袋相互作用。核磁共振光谱观察到与Kyl多肽结合时的化学位移涉及许多EphA4残基，这与广泛的相互作用和可能的受体构象变化一致。此外，系统地替换KYL和VTM的12个氨基酸中的每一个，确定对EphA4结合至关重要的残基。这些多肽在细胞培养介质中表现出较长的半衰期，与EphA4具有良好的结合亲和力和选择性，是研究调节EphA4功能的极佳工具。
The EphA4 receptor tyrosine kinase interacts with ephrin ligands to regulate many processes, ranging from axon guidance and nerve regeneration to cancer malignancy. Thus, antagonists that inhibit ephrin binding to EphA4 could be useful for a variety of research and therapeutic applications. Here we characterize the binding features of three antagonistic peptides (KYL, APY and VTM) that selectively target EphA4 among the Eph receptors. Isothermal titration calorimetry analysis demonstrates that all three peptides bind to the ephrin-binding domain of EphA4 with low micromolar affinity. Furthermore, the effects of a series of EphA4 mutations suggest that the peptides interact in different ways with the ephrin-binding pocket of EphA4. Chemical shifts observed by NMR spectroscopy upon binding of the KYL peptide involve many EphA4 residues, consistent with extensive interactions and possibly receptor conformational changes. Additionally, systematic replacement of each of the 12 amino acids of KYL and VTM identify the residues critical for EphA4 binding. The peptides exhibit a long half-life in cell culture medium, which with their substantial binding affinity and selectivity for EphA4 makes them excellent research tools to modulate EphA4 function.